Several procedures are proposed below and the
analyst must choose that which seems the most appropriate for a given extract
and the specific aim of the research. The separation of neutral lipids is only a
part of complex recipes which are described to separate all kinds of lipids, the
analyst may thus choose only the part adapted for his problem in running the
procedure from the beginning to the specific step.

First procedure

The same silica gel column as described before
for the separation of all lipid classes may be adapted for the separation of the different
neutral lipid fractions of a complex lipid extract. The step-elution is made
according to Ingalls ST et al. (J Chromatogr 1993, 619, 9).

Apparatus:

The same as previously described (general
procedure): small glass columns or SPE cartridges.

Prepare the column as previously described but
with isooctane instead of chloroform.
Wash the column with 2 ml of isooctane/ethyl acetate (80/1, v/v) and transfer the lipid
sample dissolved in 0.5 ml of the same solvent. Wash the vial with another 0.5 ml and pour
on the column.
1- Add 5 ml of the same solvent, this will elute cholesterol esters.
2- Add 5 ml of isooctane/ethyl acetate (20/1, v/v), this will elute triglycerides.
3- Rinse the lipid vial with 2 x 0.5 ml of isooctane/ethyl acetate (75/25, v/v) and pour
on the column. Add 5 ml of the same solvent mixture, this will elute cholesterol and
diacylglycerols.
4- Add 5 ml of isooctane/ethyl acetate/acetic acid (75/25/2, v/v), this will elute the
free fatty acids.
5- After rinsing the lipid vial with 2 x 0.5 ml methanol and pouring the solution on the
column, phospholipids can be eluted with 20 ml methanol.

Comments:

1- The purification of ceramides is efficiently
processed by elution with 5 ml of chloroform/methanol (95/5, v/v) after the elution of the
other neutral lipids with pure chloroform. The acylglycosylceramides (and some monogalactosyl diglycerides) are also present in the ceramide fraction. The glycolipids are
eluted as previously described or more efficiently with 10 ml of chloroform/acetone (1/1,
v/v) followed by 10 ml pure acetone.

2- To prepare a pure fatty acid fraction, the silica column is first equilibrated with
isooctane/ethyl acetate (75/25, v/v), the sample is applied to the column in the same
solvent. True neutral lipids are eluted by 5 ml of the same mixture and free fatty acids
are eluted by 5 ml of isooctane/ethyl acetate/acetic acid (75/25/2, v/v).

3- When analyses of low (trace) levels of fatty acids or other compounds are planned after
neutral lipid fractionation, it is recommended to wash the silica gel before use by 3
successive suspensions and decantation in pure methanol followed by desiccation under
vacuum lands drying at 110°C. Keep the washed silica gel in a tightly closed vessel.

Second procedure

An aminopropyl cartridge has been proposed with
success to fractionate complex lipid extracts into various neutral lipids and
sphingolipid classes (Bodennec J et al. J Lipid Res 2000, 41, 1524). The
following procedure may be used to efficiently separate ceramides from the other
neutral lipids (except monoglycerides), fatty acids but also complex sphingolipids
(sphingomyelin and glycosphingolipids). A preliminary alkaline treatment
removing glycerolipids is useful if a pure ceramide fraction (and a pure
glycosphingolipid fraction) must be prepared.

Another procedure using the same
solvents and column has been described for neutral lipids (Giacometti
J et al., J Chromatogr A 2002, 976, 47). In that work, the lipids are
loaded as chloroform solution, steryl esters are eluted with 5 ml of hexane,
triglycerides with 5 ml of hexane/methanol/chloroform (88/10/2, v/v), sterols
with 5
ml of hexane/ethyl acetate (5/95, v/v), diglycerides with 5 ml of hexane/ethyl
acetate (15/85, v/v), and monoglycerides with 5 ml of chloroform/methanol (2/1,
v/v).

An efficient procedure to isolate in pure form
some groups of neutral lipids is described below. The fractions may be further analyzed by
thin-layer chromatography or HPLC.

Reagents:

Silica gel 60 (230-400 mesh)
chloroform, hexane and diethyl ether

Procedure:

The neutral lipid fraction is first isolated
according to the general procedure on a silica gel but after elution with acidified
chloroform.
The evaporated lipid extract is dissolved in hexane and poured on the column previously
equilibrated in the same solvent.
- elute with 3 volumes of hexane, this purifies hydrocarbons including squalene
- elute with 6 volumes of hexane/diethyl ether (99/1, v/v), this purifies sterol esters,
fatty acid methyl esters and waxes
- elute with 5 volumes of hexane/diethyl ether (95/5, v/v), this purifies triglycerides,
alkyl and alkenyl acylglycerols and tocopherols
- elute with 5 volumes of hexane/diethyl ether (92/8, v/v), this purifies free fatty
acids and fatty alcohols
- elute with 8 volumes of hexane/diethyl ether (85/15, v/v), this purifies cholesterol
and a part of the diacylglycerols
- elute with 5 volumes of pure diethyl ether, this purifies monoacylglycerols and the
rest of the diacylglycerols.
In several laboratories this technique was used to prepare hundred mg of lipids from
natural sources. Although peaks may contain more than one compound, the major neutral
lipid classes can be separated on silicic acid columns and later, purified by TLC. We give
below the elution profile obtained with a column filled with 20 g of adsorbent
(dimensions: 1 x 40 cm), 10 ml fractions were collected.

A method utilizing aminopropyl bonded phase
(Bond Elut, Analytichem International) columns has been proposed to separate lipid
mixtures into individual classes of neutral lipids (free fatty acids, cholesterol esters,
triglycerides, diglycerides, monoglycerides and cholesterol) and one phospholipid class
(Kaluzny MA et al J Lipid Res 1985, 26, 135).
Although this method appeared difficult to manage in our hands, it may be adapted to
various materials with some effort but affords rapid and convenient separations of several
samples in one process with the described collector rack.
A modification of the Kaluzny procedure adapted more precisely for ceramides and
glycosphingolipids (in the presence of neutral lipids) is reported above (second
procedure).